Am J Transl Res 2015;7(3):430-444

www.ajtr.org /ISSN:1943-8141/AJTR0006360

Original Article
Pulsed electromagnetic field improves postnatal
neovascularization in response to hindlimb ischemia
Rui-Lin Li1*, Jing-Juan Huang1*, Yi-Qin Shi2*, An Hu3, Zhao-Yang Lu1, Liang Weng1, Shen-Qi Wang1, Yi-Peng
Han1, Lan Zhang2, Chang-Ning Hao2, Jun-Li Duan1
1
Department of Gerontology, Xinhua Hospital, Shanghai Jiaotong University, Kongjiang Road 1665, Shanghai
200092, China; 2Department of Vascular Surgery, Ren Ji Hospital, Shanghai Jiaotong University School of
Medicine, Dongfang Road 1630, Shanghai 200127, China; 3Department of Otolaryngology, Gong li Hospital,
Miaopu Road 219, Shanghai 200135, China. *Equal contributors.
Received January 26, 2014; Accepted February 25, 2015; Epub March 15, 2015; Published March 30, 2015

Abstract: Pulsed electromagnetic fields (PEMF) have been shown to promote proliferation and regeneration in the
damaged tissue. Here, we examined whether PEMF therapy improved postnatal neovascularization using murine
model of hindlimb ischemia, and the underlying cellular/molecular mechanisms were further investigated. Hindlimb
ischemia was induced by unilateral femoral artery resection using 6-8 week-old male C57BL6 mice. Then, mice
were exposed to extracorporeal PEMF therapy (4 cycles, 8min/cycle, 30 ± 3 Hz, 5 mT) every day until day 14. Our
data demonstrated that PEMF therapy significantly accelerated wound healing, decreased prevalence of gangrene
and increased postnatal neovascularization. Moreover, the levels of vascular endothelial growth factor (VEGF), en-
dothelial nitric oxide synthase (eNOS) and Akt phosphorylation in ischemic muscles were markedly enhanced follow-
ing PEMF therapy. In vitro, PEMF inhibited the process of hypoxia-induced apoptosis and augmented tube formation,
migration and proliferative capacities of human umbilical vein endothelial cells (HUVECs). Additionally, PEMF expo-
sure increased VEGF secretion, as well as the eNOS and Akt phosphorylation, and these benefits could be blocked
by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. In conclusion, our data indicated that PEMF therapy
enhanced ischemia-mediated angiogenesis, through up-regulating VEGF expression and activating the PI3K-Akt-
eNOS pathway. Therefore, PEMF should be a valuable treatment for the patients with critical limb ischemia.

Keywords: Pulsed electromagnetic fields (PEMF), angiogenesis, hindlimb ischemia, VEGF, eNOS

Introduction Pulsed electromagnetic field (PEMF) is a non-

invasive and non-pharmacological intervention
Peripheral artery disease (PAD) is an increasing and reported to repair damaged tissue [4-8].
clinical scenario defined as the impairment of Several clinical studies have demonstrated th-
blood flow to extremities, which is most com- at PEMF effectively alleviate traumatic pain, tis-
mon caused by atherosclerotic progression. sue swell and promote wound healing [9]. PEMF
The exacerbation of limb ischemia, which is also accelerates the fusion of fractured bones
associated with aging, black race/ethnicity, [10], as well as contributes to neuronal regen-
cigarette, diabetes, hypertension and hyper- eration and differentiation [11, 12]. Recent
cholesterolemia, would lead to critical limb studies indicated that PEMF stimulated angio-
genesis and improved microcirculation in
ischemia (CLI) [1, 2]. It is reported that the inci-
patients with diabetes [13-15]. However the
dence of limb ischemia is 1.5 cases per 10000
detailed mechanism remains modest unders-
persons per year in USA [3]. Despite achieve-
tood.
ments of therapeutic strategies against PAD,
clinicians are still facing increasing challenges We hypothesized that PEMF can promote PAD
of refractory CLI. Recently, therapeutic angio- through accelerating angiogenesis. In the pres-
genesis have been applied to treat no-option ent study, we introduced PEMF treatment to a
patients with CLI. well-established mouse model of hindlimb isch-
 PEMF improves angiogenesis in ischemic hind limb mice

Figure 1. PEMF reduces the occurrence of necrosis or skin ulcers after hindlimb ischemia. At day 14 after induction
of ischemia, the general condition of hindlimb was photographed (A), and, the ischemia was scored according to a
recognized evaluation standards (B). Values are mean ± SEM; n = 6, N.S. means no significant difference, *means
P < 0.05, **means P < 0.01.

emia and investigated the role PEMF in isch- hindlimb ischemia was surgically induced and
emia-mediated angiogenesis. PEMF was performed among groups as previ-
ously described [8, 16]. Wound healing and the
Materials and methods gangrene occurrence was observed during the
next 14 days after surgery. The blood perfusion
Animals was detected indirectly by the temperature of
the limbs on postoperative day 0 and day 14
Male, 8-week-old C57BL/6 mice (18-20 g) were [17]. The rapid development of the infrared
purchased from Shanghai SLAC Laboratory detection technology [18-20] promote the ther-
Animal Co. Ltd (Shanghai, China) and acclimat- mal infrared image to be a very useful noncon-
ed in an environmentally controlled breeding tact temperature measurement method to get
room for 5 days prior to the experiments. All the the temperature image of animals. This thermal
mice had free access to food/water supplies infrared image method is used to determine
and were fasting overnight before experiments. the skin temperature of our studied mice.
The study was approved by the Animal Ethics Animals were euthanized after finishing infra-
Committee of the Shanghai Jiao Tong University red spectrum imaging on postoperative day 14.
and conducted in strict accordance with the The thigh adductor muscle was immersed in
recommendations in the Guide for the Care and 4% formaldehyde and embedded in paraffin for
Use of Laboratory Animals of the National immunohistochemistry analysis. For fluores-
Institutes of Health. All surgeries were per- cent immunohistochemistry, samples were
formed under anesthesia, and all efforts were snapped frozen in liquid nitrogen and stored at
made to minimize suffering. -80°C until analysis.

Animal study protocol and sampling Hindlimb ischemia model

Male C57BL/6 mice were randomly divided into Unilateral hindlimb ischemia was surgically
4 groups as follow: (1) skin cut without artery induced as previously described [16]. Mice
excision (sham group, n = 6), (2) sham opera- were anesthetized with 4% chloral hydrate (40
tion but treated with PEMF (sham + PEMF mg/kg, i.p.) under sterile surgical conditions.
group, n = 6), (3) left femoral artery resection We performed a longitudinal incision in the left
without PEMF (ischemia group, n = 6), (4) left hindlimb; the femoral artery was completely
femoral artery resection treated with PEMF excised from its proximal origin to the point
(ischemia + PEMF group, n = 6). Unilateral where it bifurcated into the popliteal and saphe-

431 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 2. PEMF improves the blood flow of the ischemic hindlimb. Infrared spectrum imaging assay was performed on day 0 and day 14 after the hindlimb ischemia
operation (A and C). The data was analyzed and illustrated as an ischemic/normal hindlimb temperature ratio (B and D). Values are mean ± SEM; n = 6 , N.S. means
no significant difference,*means P < 0.05, **means P < 0.01.

432 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 3. Effects of PEMF on capillary density in ischemic hindlimb. CD31-positive cells were stained in the left thigh
adductor muscle at day 14 after induction of ischemia (A) Capillary density was identified with capillary per field
(× 400 magnification, (B) Values are mean ± SEM; n = 6, N.S. means no significant difference, *means P < 0.05,
**means P < 0.01. Scale bar indicated 20 μm.

nous arteries including all branches of the fem- lyzer (Prism-DS 50137, FLIR Systems) to evalu-
oral artery. ate the blood perfusion [22, 23]. Low tempera-
ture was displayed as dark-to-purple, whereas
PEMF treatment high temperature was displayed as red-to-
PEMF was generated by a commercially avail- white. We performed infrared radiation image
able healing device purchased from Biomobie detection over the same region of interest
Regenerative Medicine Technology (Shanghai, immediately after surgery (day 0) and day 14,
China). Fields were asymmetric and consisted temperature values was computed from histo-
of 4.5 ms pulses at 30 ± 3 Hz, with a magnetic grams of the colored pixels. To minimize varia-
flux density increasing from 0 to 5 mT in 400 μs tions due to ambient light, the TIRI data was
as described before [8]. The mice among PEMF expressed as the ischemic/normal limb tem-
groups were housed in custom-designed cages perature ratio.
and exposed to active PEMF for 32 minutes (4
cycles, 8 minutes per cycle) per day until day 14 Immunofluorescence analysis
after surgery.
Immunofluorescence staining for capillary den-
Hindlimb ischemic score sity was performed as described previously
[24]. Briefly, 14 days after surgery, mice were
The ischemia score was assessed as described sacrificed and the thigh adductor muscles were
previously [21]. Briefly, mice were investigated fixed in 4% paraformaldehyde for 24 h. Then,
14 days after surgery and assigned one of the samples were washed, dehydrated in a graded
following scores: 0, no necrosis or skin ulcers ethanol series and embedded in paraffin. 5
occurred; 1, skin ulcers; 2, below ankle ampu- µm-sections were cut and blocked with PBS
tation; and 3, above ankle amputation. Two containing 1% BSA and 0.1% Tween 20. The
researchers evaluated the ischemia score in a sections were incubated with goat anti-CD31
blinded manner and the average scores for antibody (5 µg/ml, BD Biosciences, Franklin
each animal were used for quantitative ana-
Lakes, NJ) at 4°C overnight. After washing with
lysis.
PBS containing 0.1% Tween 20, the sections
Thermal infrared imaging (TIRI) analysis were incubated with Alexa 488-conjugated
anti-rat IgG goat polyclonal antibodies (250-
The skin temperatures of both hind limbs were fold dilution, Invitrogen, Carlsbad, CA) at room
measured by the thermal infrared imaging ana- temperature for 30 minutes in a dark room.

433 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 4. PEMF promotes the expression of VEGF, p-Akt and p-eNOS in vivo. Quantitative analysis of protein content of VEGF, Akt, phosphorylated Akt (p-Akt), eNOS
and phosphorylated eNOS (p-eNOS) were analyzed 14 days after operation. Data of Western blotting was represented as fold of sham. Values are mean ± SEM; n
= 6, N.S. means no significant difference, **means P < 0.01.

434 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 5. PEMF accelerates the proliferation of HUVECs without significant toxicity. HUVECs were stimulated by
PEMF for 1-4 cycles and the proliferation was analyzed by Cell Count Kit-8 (CCK-8) in several time point as indicated
(A). The cell toxicity was also checked (B). Values are mean ± SEM; n = 4, N.S. means no significant difference,
*means P < 0.05, **means P < 0.01, vs. control group.

Capillary density was determined by counting bance of the reaction system at 0.5 h, 1 h, 2 h,
of 10 randomly selected fields and is expressed and 4 h was measured at 450nm wavelength.
as numbers of capillary/field (× 400 magnifica- Additionally, HUVECs (5 × 104/mL) suspended
tion) [25, 26]. in DMEM (100 µL) were operated followed by
the above-mentioned protocol for indicating
Cell culture the toxicity of PEMF.

Human umbilical vein endothelial cells (HUVEC; Apoptosis analysis

ATCC, Cat. CRL1730) were purchased from cul-
tured in DMEM (low-glucose plus 10% FBS) and HUVECs were seeded on sterile cover glasses
supplemented with 100 U/ml penicillin and placed in the 6-well plates and cultured in
100 U/ml streptomycin at 37°C under a humidi- serum-free DMEM for 48 h. HUVECs were then
fied 95%: 5% (v/v) mixture of air and CO2. treated with PEMF as indicated (1-4 cycles
HUVECs were reseeded into plates overnight with/without L-NAME or LY294002 treatment)
and stimulated at indicated time point flowing for consecutive 3 days. Next, cells were fixed
PEMF exposure 1-4 cycles/day for 3 days, 30 ± with 4% paraformaldehyde, washed twice with
3 Hz, 5 mT). Supernatant and cell lysates were PBS and stained with Hoechst 33258 staining
collected for biological analysis. solution according to the manufacturer instruc-
tions (Beyotime). Hoechst-stained pyknotic nu-
Reagents clei were counted as percentage of 100 cells in
each well (× 400 magnification). Each group
All drugs were obtained from Beyotime, Jiangsu, was studied at least in triplicate [27].
China. L-NAME, an eNOS inhibitor, was dis-
solved in PBS and used at a final concentration Migration assay
of 50 μM. LY294002, a phosphoinositide 3-
kinase (PI3K) inhibitor, was dissolved in DMSO Migration assay was performed using 24-well
and used at a final concentration of 20 μM. Boyden Transwell chambers (Corning, Camb-
ridge, MA) with 6.5-mm-diameter polycarbon-
CCK-8 assay ate filters (8-µm pore size) [28]. Briefly, 600 µL
DMEM containing 10% FBS was added into the
Cell growth was analyzed using a WST-8 Cell lower compartment, HUVECs (3 × 105/mL) sus-
Counting Kit-8 (CCK-8 kit, Beyotime). HUVECs pended in 100 µl DMEM (no serum) were seed-
(2 × 104/mL) were seeded in 96-well plates ed in upper compartment of the Transwell
with 100 μL DMEM (with 10% FBS) and exposed chambers. Cells were then treated with PEMF
to PEMF for 1-4 cycles The HUVECs were then for different cycles as indicated with/without
incubated at 37°C for 4 h after the CCK-8 solu- L-NAME or LY294002. After 8 h incubation,
tion (10 µL) was added to each well. The absor- upper compartments were removed, whereas

435 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 6. PEMF protects the apop-

tosis of HUVECs in vivo. Represen-
tative images of Hoechst33258
Staining in HUVECs were stimu-
lated by PEMF for 1-4 cycles,
and 4 cycles group treated with
L-NAME or LY294002 and stained
by Hoechst33258 (A) Quantita-
tive analysis was represented as
the apoptotic cells in the total
cells per field (B) Values are mean
± SEM; n = 4, N.S. means no sig-
nificant difference, ** means P <
0.01, vs. control; ## means P <
0.01, vs. 4 cycles group. Scale bar
indicated 20 μm.

the cells that migrated through the membrane Tube formation assay
to the underside were fixed with cold 4% para-
formaldehyde and stained with 0.1% crystal The tube formation assay was performed as
violet. Cell numbers were counted in 5 sepa- described previously [29]. Briefly, Matrigel-
rate fields using light microscopy at × 200 Matrix (BD Biosciences) was added in the well
magnification. of a 48-well cell culture plate and HUVECs (5 ×
105/mL) suspended in 50 µl DMEM (with 10%
Wound healing assay FBS) were seeded and treated with PEMF. After
6-8 h incubation, images were acquired under
HUVECs were cultured and grown to 100% con- a fluorescent microscope (IX-71; Olympus,
fluence in 6-well plates. A clear area was then Tokyo, Japan) with 12.8 M pixel recording digi-
scraped in the monolayer with a 200 µl pipette tal color cooled camera (DP72; Olympus). The
tip. After washed by PBS for three times，HUVECs tube formation was calculated as fold of con-
were cultured with serum-free DMEM, treated trol. Each experiment was repeated 4 times
with PEMF for different cycles as indicated. under similar conditions, images of tube mor-
Migrated cells into wounded areas was evalu- phology were taken and tube lengths were cal-
ated with a phase contrast microscope and culated under × 100 magnification.
photographed 24 h and 48 h later (× 100 mag-
nification). The healing rate was quantified with Western blotting
measurements of the narrowing down gap size
by the formula: 100% - (width 24 h or 48 h/ Equal amounts of total protein from the extracts
width 0 h) × 100%. Three different areas in of thigh muscle and HUVECs were resolved in
each assay were randomly chosen. SDS 10% polyacrylamide gel and transferred to

436 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 7. PEMF promotes the migration

of HUVECs. HUVECs were plated in tran-
swell and stimulated by PEMF for 1-4
cycles, and 4 cycles group treated with
L-NAME or LY294002. Migrated cells
were stained (A) and quantitative anal-
ysis of migrated cells was represented
as fold of control (B). Wound healing
analyze was performed 24 h and 48 h
(C and D). Values are mean ± SEM; n =
4, N.S. means no significant difference,
*means P < 0.05, **means P < 0.01,
vs. control; ##means P < 0.01, vs. 4 cy-
cles group. Scale bar indicated 50 μm.

nitrocellulose membranes for Western blotting applied GAPDH as an internal control to stan-
as described previously [16, 30]. The primary dardize to the protein quantity.
antibodies used were as follows: anti-eNOS
(Sigma, St. Louis, MO), anti-phosphor-eNOS Statistical analysis
(Sigma), anti-VEGF (Proteintech, Chicago, IL),
anti-Akt, anti-phosphor-Akt (p-Akt) and anti- One-way ANOVA analysis of variance with the
GAPDH (Cell Signaling Technology, Beverly, post-hoc Tukey test was applied for multiple
MA). Positive signals were visualized with a Flu- comparisons. SPSS software version 17.0 (SP-
orChem E data system (Cell Biosciences, Santa SS Inc., Chicago, IL) was used. All experiments
Clara, CA) and quantified by densitometry using were performed at least in triplicate. A value of
Quantity One 4.52 (Bio-Rad, Hercules, CA). We P < 0.05 was considered significant.

437 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 8. PEMF contributes to

tube formation. Representa-
tive images of tube formation
in HUVECs by stimulated PEMF
for 1-4 cycles, and 4 cycles
group treated with L-NAME or
LY294002 (A) and quantitative
analysis of tube length were
represented as fold of control
(× 100 magnification, B). Val-
ues are mean ± SEM; n = 4,
*means P < 0.05, **means
P < 0.01 vs. control; ##means
P < 0.01, vs. 4 cycles group.
Scale bar indicated 100 μm.

Results therapy, the temperature ratio of PEMF-treated

mice was dramatically increased in comparison
PEMF reduced the prevalence of limb necrosis with PEMF-free mice ischemic groups, suggest-
and ischemic ulcers ed that PMEF accelerate the blood flow recov-
ery of hindlimb ischemia (Figure 2B and 2C). To
To investigate whether PEMF promoted wound confirm our observation in microcirculation
healing, we observed the general recovery of level, we preformed anti-CD31 immunofluores-
ischemic limbs and recording the occurrence of cence stainings to quantify the capillary densi-
skin ulcers and gangrene 14 days after hindlimb ty. We demonstrated that capillary density was
ischemia (Figure 1A). We found that the isch- increased in PEMF-treated mice compared with
emic score among PEMF-treated mice was sig- non-treated ones in ischemic group 14 days
nificantly lower than that in non-treated ones after operation (Figure 3).
either between operation groups (P < 0.01) or
sham groups (P < 0.05). These results indicat- PEMF induced the expressions of angiogenic
ed that PEMF could effectively accelerate skin factors in vivo
wound healing and rescue ischemic limbs
(Figure 1B). To further examine the underlying mechanisms
of PEMF-induced angiogenesis, we evaluated
PEMF improved angiogenesis in ischemic area the expressions of several angiogenic factors.
Although no difference of VEGF level could be
The representative photographs of infrared found between 2 sham groups, PEMF treat-
spectrum in day 0 and 14 were illustrated in ment significantly increased VEGF expression
Figure 2A and 2C. Fourteen days after PEMF in ischemic groups (Figure 4A). Moreover, the

438 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

phosphorylation levels of Akt (p-Akt, Figure 4B) PEMF enhanced angiogenic factor expressions
as well as eNOS (p-eNOS, Figure 4C) contained in vitro
in the ischemic muscle were also up-regulated
at postoperative day 14 in response to 4-cycle PEMF promoted VEGF released from HUVECs in
PEMF exposure. a dose dependent manner (Figure 9A). Fur-
thermore, the phosphorylation of Akt and eNOS
PEMF accelerated the proliferation of HUVECs in HUVECs was also upregulated in response to
PEMF following a dose dependent manner
HUVECs were stimulated by PEMF in different (Figure 9B and 9C). The PEMF’s benefits can be
duration (1-4 cycles, 8 min per cycle, 30 ± 3 Hz, neutralized by administration of L-NAME and
5 mT), cell growth was analyzed by CCK-8 assay LY294002 (Figure 10). These results indicated
in several time point as indicated. We found that PEMF triggered angiogenesis via activa-
that PEMF promoted cell growth was promoted tion of Akt-eNOS-VEGF axis.
in treated groups from 1 h after the reaction to
compare with the control group and became Discussion
significantly higher 4 h later (Figure 5A). The
HUVECs’ proliferation was enhanced by PEMF In present study, we demonstrated that PEMF
in a dose-dependent manner. During the pro- exposure improved ischemia-induced angio-
cess, there wasn’t any system toxicity exposed genesis through enhancing endothelial prolif-
to the PEMF treatment (Figure 5B). This in vitro eration, migration, survival and secretion via
experiment further proved that PEMF could acting on the Akt-eNOS-VEGF pathway of endo-
improve angiogenesis effectively. thelial cells.

PEMF inhibited serum free-induced HUVEC Physiotherapy-mediated regenerative medicine

apoptosis takes several advantages compared with cell /
pharmaco-mediated therapies. Extracorporeal
As shown in Figure 6A, Hoechst staining was PEMF was reported to enhance osteanagene-
performed to determine the proportion of apop- sis [10], skin rapture healing [9] and neuronal
totic cells by manually counting pyknotic nuclei. regeneration [11, 12], suggesting its regenera-
The proportions of apoptotic cells were dramat- tive potency. Previously, we reported that PEMF
ically decreased in the HUVECs that stimulated preserved heart systolic function and rescued
by PEMF for 3-4 cycles compared with others. apoptotic cardiomyocytes using mice model of
Moreover, this benefit could be markedly atten- myocardial infarction [8]. Present study demon-
uated using eNOS inhibitor L-NAME or PI3k strated that PEMF therapy increased tissue
inhibitor LY-294002 (Figure 6B). repairmen and proliferation via promoting capil-
lary endothelial growth and skin temperature of
PEMF promotes migration and tube formation the ischemic hindlimb, even in sham operation
of HUVECs group, suggesting that the PEMF-induced accel-
erated wound healing might due to increased
Two kinds of migration assay were performed blood perfusion to the ischemic limb.
to evaluate endothelial migration ability in
response to PEMF treatment. In transwell To further explore the mechanism of PEMF-
migration assay, both 3 cycle- and 4 cycle-treat- mediated neovascularization, we studied PE-
ed groups showed significantly more migrated MF-induced endothelial biological modification
HUVECs than control group (Figure 7A and 7B). in vitro. We clarified that PEMF improved multi-
PEMF also accelerated scratch wound closure ple endothelial functions including migration,
in comparison with control group following a proliferation, tube formation and VEGF secre-
dose dependent manner (Figure 7C and 7D) tion, suggesting the PEMF-induced therapeutic
and these benefits could be blocked by L-NAME potency targeted on pre-existing endothelial
and LY294002. In tube formation assay, the vasculature. On the other hand, we previously
HUVECs-formed micro-tubes were lengthened demonstrated PEMF exposure promoted sca-
in response to PEMF exposure following a dose 1+flk-1+ endothelial progenitor cells (EPCs)
dependent manner via Akt-eNOS axis (Figure recruitment to the ischemic heart using mice
8). model of myocardial infarction [8]. Goto and

439 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 9. PEMF promotes the expression of VEGF, p-Akt and p-eNOS in vitro. Quantitative analysis of protein content of VEGF, Akt, p-Akt, eNOS and p-eNOS in HUVECs
stimulated by PEMF for 1-4 cycles. Data of Western blotting were represented as fold of control. Values are mean ± SEM; n = 4, *means P < 0.05, **means P <
0.01, vs. control.

440 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

441 Am J Transl Res 2015;7(3):430-444

 PEMF improves angiogenesis in ischemic hind limb mice

Figure 10. Angiogenic factors of PEMF was eliminated by eNOS inhibitor or PI3K inhibitor. Quantitative analysis of
protein content of VEGF, Akt, p-Akt, eNOS and p-eNOS in HUVECs stimulated by PEMF for 4 cycles treated with L-
NAME (A) or LY294002 (B-D). Data of Western blotting were represented as fold of control. Values are mean ± SEM;
n = 4, **means P < 0.01.

colleagues also reported that PEMF stimulation esis. These findings highlighted the powerful
up-regulated the expressions of angiopoietin-2 therapeutic potential of PEMF for a safe and
and fibroblastic growth factor-2 in bone mar- simple healing response with better blood per-
row, suggesting PEMF could promote the regen- fusion in patients with critical limb ischemia.
erative capacity of myeloid-derived cells (such
as EPCs) in damaged tissue when recruited [4]. Acknowledgements
Taken together, PEMF triggered postnatal neo-
This work was supported by the China National
vascularization through acting on both pre-
Natural Science Foundation (11374213) and
existing endothelial cells and circulating EPCs.
Foundation of National Lab for Infrared Physics
To the best of our knowledge, we first revealed (200901).
the molecular mechanism of PEMF-mediated Disclosure of conflict of interest
angiogenesis in ischemic thigh. The PEMF-
induced therapeutic benefits were abolished None.
when either PI3K inhibitor (LY294002) or eNOS
deactivator (L-NAME) was administrated to the Address correspondence to: Dr. Jun-Li Duan, De-
culture dish. Our study provides convinced data partment of Gerontology, Xinhua Hospital, Shanghai
that PEMF stimulated Akt-eNOS-VEGF axis Jiaotong University, Kongjiang Road 1665, Shanghai
using PI3K and eNOS pathway inhibitors in 200092, China. E-mail: [email protected]; Dr.
vitro. VGEF and nitric oxide (NO) appear to Chang-Ning Hao or Lan Zhang, Department of
mediate distinct, but interdependent, pathways Vascular Surgery, Ren Ji Hospital, Shanghai Jiaotong
of angiogenesis in response to ischemic stress. University School of Medicine, Dongfang Road
VEGF increases the permeability and prolifera- 1630, Shanghai 200127, China. Tel: + 86-21-2507-
tion of endothelial cells so that triggers endo- 7715; Fax: + 86-21-6549-3951. E-mail: gilbertha-
thelial sprouting [31]; nitric oxide, on the other [email protected] (CNH); [email protected]
hand, induces mature endothelial markers (LZ)
expression (such as VE-cadherin) [32]. Our data
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